The present invention relates to a method of processing recombinant procollagen.
Collagens are the main proteins responsible for the structural integrity of vertebrates and many other multicellular organisms. Type I collagen represents the prototypical fibrillar collagen and is the major collagen type in most tissues, including bone, tendon, skin, aorta, and lung. Type I collagen fibers provide for great tensile strength and limited extensibility.
Collagen provides biomaterials with characteristics necessary for a myriad of applications including pharmaceutical (haemostatic compresses, sponges, healing dressings), medical (prostheses such as cardiac valves, tendons and ligaments, skin substitutes, filling agents), odontological (gum implants) and cosmetic (additive, anti-wrinkling agent, microcontainer for perfumed substances). The collagen-based products manufactured in all of the aforementioned markets require vast amounts of raw collagen materials for their production.
The conformation and most of the properties of native collagen are determined by the triple helix domain which composes more than 95% of the molecule. This domain consists of three alpha chains, each containing approximately 1,000 amino acids, wrapped in a rope-like fashion to form a tight, triple helix structure. The triple helix is wound in such a way that peptide bonds linking adjacent amino acids are buried within the interior of the molecule, such that the collagen molecules are resistant to attack by proteases, such as pepsin.
In all of the fibrillar collagen molecules, the three polypeptide chains are constructed from repeating Gly-X-Y triplets, where X and Y can be any amino acid but are frequently the imino acids proline and hydroxyproline. An important feature of fibril-forming collagens is that they are synthesized as precursor procollagens containing globular N- and C-terminal extension propeptides. The triconstituent polypeptide chains are assembled within the rough endoplasmic reticulum to form procollagen. As the polypeptide chain is co-translationally translocated across the membrane of the endoplasmic reticulum, prolyl-4-hydroxylase (P4H)-dependent hydroxylation of proline and lysine residues occurs within the Gly-X-Y repeat region. The stability of the final triple-helical structure of collagen is highly dependent on the P4H-mediated hydroxylation of collagen chains. Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity (Wang et al, 2002, Matrix Biol. November; 21(7):559-66). A single human enzyme, Lysyl hydroxylase 3 (LH3) can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation (Wang et al, 2002, Matrix Biol. November; 21(7):559-66). Once the polypeptide chain is fully translocated into the lumen of the endoplasmic reticulum the three pro-alpha chains associate via their C-propeptides to form a trimeric molecule where the Gly-X-Y repeat region forms a nucleation point at its C-terminal end, ensuring correct alignment of the chains. The Gly-X-Y region then folds in a C-to-N direction to form a triple helix (J. Khoshnoodi. et. al, J. Biol. Chem. 281, 38117-38121, 2006)
The C-propeptides (and to a lesser extent the N-propeptides) keep the procollagen soluble during its passage out of the cell (Bulleid et al., 2000, Biochem Soc Trans; 28(4):350-3). Following or during secretion of procollagen molecules into the extracellular matrix, propeptides are typically removed by procollagen N- and C-proteinases, thereby triggering spontaneous self-assembly of collagen molecules into fibrils (Hulmes, 2002, J Struct Biol. January-February; 137(1-2):2-10). Removal of the propeptides by procollagen N- and C-proteinases lowers the solubility of procollagen by >10000-fold and is necessary to initiate the self-assembly of collagen into fibers at 37° C. Crucial to this assembly process are the short telopeptides which are the nontriple-helical remnants of the N- and C-terminal propeptides remaining after digestion with N/C proteinases. These peptides act to ensure correct covalent registration of the collagen molecules within the fibril structure and lower the critical concentration required for self-assembly (Bulleid et al., 2000, Biochem Soc Trans; 28(4):350-3) through their crosslinkable aldehydes.
Native collagen is generally present in connective tissue as telopeptide-containing collagen molecules packed side by side in the form of fibrils. Each longitudinal course is composed of molecules aligned in end-to-end dispositions with slight longitudinal spaces staggered relative to the next successive laterally adjacent longitudinal course. In this way, gaps are generated between facing end regions of successive molecules in a given longitudinal course and bound by the staggered sides of the molecules in the parallel longitudinal courses laterally adjacent thereto.
Dispersal and solubilization of native animal collagen can be achieved using various proteolytic enzymes which disrupt the intermolecular bonds and remove the immunogenic non-helical telopeptides without affecting the basic, rigid triple-helical structure which imparts the desired characteristics of collagen (see U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods for preparing purified soluble collagen). The resulting soluble atelocollagen can be subsequently purified by repeated precipitation at low pH and high ionic strength, followed by washing and re-solublization at low pH. Nevertheless, the soluble preparation is typically contaminated with crosslinked collagen chains which decrease the homogeneity of the protein preparation.
The use of animal-derived collagen is problematic due to the possible risks of contamination by non-conventional infectious agents. While the risks raised by bacterial or viral contamination can be fully controlled, prions are less containable and present considerable health risks. These infectious agents which appear to have a protein-like nature, are involved in the development of degenerative animal encephalopathy (sheep trembling disease, bovine spongiform encephalopathy) and human encephalopathy (Creutzfeld-Jacob disease, Gerstmann-Straussler syndrome, and kuru disease). Due to the lengthy time before onset of the disease, formal controls are difficult to conduct.
Plants expressing collagen chains are known in the art, see for example, WO06035442A3; Merle et al., FEBS Lett. 2002 Mar. 27; 515(1-3):114-8. PMID: 11943205; and Ruggiero et al., 2000, FEBS Lett. 2000 Mar. 3; 469(1):132-6. PMID: 10708770; and U.S. Pat. Applications 2002/098578 and 2002/0142391 as well as U.S. Pat. No. 6,617,431.
U.S. Pat. Nos. 4,597,762, 5,670,369, 5,316,942, 5,997,895 and 5,814,328 teach processing of animal derived “insoluble collagen” with plant derived proteases such as ficin and/or papain.